CD4+Foxp3+ regulatory T (TR) cells are crucial to the maintenance of peripheral tolerance. Natural regulatory T (nTR) lymphocytes are a distinct thymus-derived lineage. A second subset of induced Foxp3+ regulatory T (iTR) cells can be generated de novo from conventional CD4+Foxp3- T cells upon antigenic stimulation in the presence of TGF-beta and IL-2. While both nTR and iTR cells are dependent on the expression of the forkhead transcription factor Foxp3 for their differentiation and suppressive action, the two populations are molecularly and functionally distinct. In both humans and in mice, the failure of TR cells to differentiate due to loss of function mutations in Foxp3 results in a lethal disease of systemic autoimmunity, lympho-proliferation and allergic dysregulation. Foxp3 mutations also result in the accumulation of TR cell precursors that have failed to differentiate into functional TR cells in tissues targeted by the autoimmune inflammatory process. These cells, which are predicted to be autoreactive, are highly proliferative and actively produce large amounts of cytokines and granzymes, and thus may contribute to disease pathology. Our recent studies on Foxp3-deficient mice revealed that the disease can be dissociated into two main components: one that is dependent on the innate immune regulator MyD88 and which involves inflammation at the mucosal surfaces in the skin, gut and lungs, and another that is MyD88-independent, manifesting as unrestrained systemic lympho- and myelo-proliferation. Whether this dichotomy reflects a division of labor between nTR and iTR cells or reflects the utilization of distinct TR cell effector molecules such as IL-10 and CTLA-4 remains unclear. Our long-term goal is to dissect the role of Foxp3-regulated pathways in the induction and maintenance of immunologic tolerance. The focus of this proposal is to identify key cellular and molecular mechanisms by which Foxp3 deficiency promotes autoimmunity and inflammation. We hypothesize that Foxp3 deficiency unleashes unrestrained activation of both the innate and the adaptive immune responses, driven by the regulatory failure of both nTR and iTR cells. Furthermore, we hypothesize that the aborted TR cell precursors seen in Foxp3 deficiency represent a unique class of tissue-specific autoimmune effector cells that contributes to disease pathogenesis and tissue damage. The proposed studies will provide fundamental insights into the pathogenesis of diseases associated with TR cell deficiency and will enable novel therapeutic approaches employing TR cell-based interventions.